抗突变耐药的ALK/ROS1双靶点选择性抑制剂的设计、合成及作用机制研究

ALK- and ROS1-oncogene fusion proteins are effective molecular targets for “personalized medicine” in non-small celll lung cancers, but the mutations in kinase domain of ALK-and ROS1-oncogene fusion proteins are responsible for ALK inhibitor resistance in these cancers. It is a medicinal chemistry campaign aimed at improving the feasibility of built selected ALK/ROS1 dual inhibitors against crizotinib-resistant mutants based on the structural conformation of mutation point in kinase domain. In our previous study, we found a 2-aminopyrimidine compound ZXN-17 which showed strong ALK and ROS1 inhibitory activity. Encouraged by previous results, aiming to G1202R ALK/G2032R ROS1 mutation points,this project will develop multiplex inhibitors of ALK- and ROS1 based on their mutation conformation by using of molecular Docking and molecular dynamics simulation. The design strategies of "mix-n-match" and "hybridization" will be adopted with ZXN-17 as lead compound. Eight kinds of molecules will be synthesized. In order to evaluate the potency and selectivity of the title compounds, various biological assays and primary safety evaluation will be carried out. This project will identify 1-3 lead compound with a "dual" ALK/ROS1inhibition profile. The mechanisms of action of lead compound will be illuminated by means of co-crystal research between lead with target protein combined with the research on the downstream signaling pathways of ALK and ROS1 targets. The project would resovle two questions. One is how to design the effective molecules against the mutated ALK and ROS1; the other is how to identify the viability, activity and mechanisms of the title compounds. As a result, this project may provide the scientific basis to the design of novel ALK/ROS1 selective dual inhibitors against crizotinib resistant mutants.

ALK及ROS1融合蛋白是非小细胞肺癌等肿瘤个性化治疗的重要分子靶点，但激酶结构域突变引起的耐药使现有药物无效。本项目旨在探索“基于激酶突变位点的立体构象构建抗突变耐药的ALK/ROS1选择性抑制剂”的可行性。以前期发现的对野生型及突变型ALK/ROS1均有较强抑制活性的2-氨基嘧啶衍生物ZXN-17为先导化合物，针对G1202R ALK/G2032R ROS1突变位点，本项目拟通过化合物与突变蛋白晶体结构的分子对接、动力学模拟手段，采用"Mix-n-match"杂合策略，构建满足突变蛋白构象要求的化合物库，合成8类目标分子，通过多水平的活性筛选及安全性评价，优选出1-3个先导化合物；开展先导物与靶标的共晶研究，结合对靶标下游信号通路的研究阐明其作用机制，解决①如何设计对突变靶标有效的分子；②如何验证其活性与机制两个关键问题，为抗突变耐药的ALK/ROS1选择性抑制剂的设计提供科学依据。

本课题严格按照申请书拟定计划开展研究，着力开展了基于2,4-二芳氨基嘧啶结构的抗突变耐药ALK/ROS1双靶点选择性抑制剂的发现、优化及分子机制研究。针对ALK的G1202R及L1196M突变位点，采用“Mix-n-match”杂合策略，设计并合成了15个系列的二芳氨基嘧啶类新化合物570余个；同时，基于变构调节设计合成了含哌啶甲酸及螺环结构的新型Type-I1/2型ALK抑制剂60个，构建了结构类型多样的化合物库，建立构效关系模型。通过酶（HTRF法）、细胞（MTT法）及在体动物（PDX模型）水平的药理活性筛选，优选出高效低毒的4个候选分子（ZX-42、ZX-137、ZSJ-1和AR-24），其抑酶活性约为1 nM，对肿瘤移植瘤的抑制率可达91.8%，优于Ceritinib；对AR-24的研究获得2020年中国药学会-以岭生物医药创新基金项目的后续资助。完成了药代动力学研究等初步安全性评价，采用基因敲除技术，构建了突变耐药模型。基于Western blot、PCR、免疫荧光染色等技术，探索目标分子对野生型及突变型ALK/ROS1及其下游信号通路蛋白的影响，结合细胞周期、细胞凋亡等相关研究，初步阐明了抗肿瘤分子机制及耐药机制。本课题不仅为ROS1/ALK 高选择性抑制剂的合理设计提供了充分的科学依据，同时发现了4个高活性候选分子，为开发抗肿瘤一类新药提供了物质基础；阐释了耐药机制，将有助于具有自主知识产权的抗ALK突变药物的发现，实现全新的突破。.本课题超额完成了申请书拟定的研究任务，学术成果丰硕。已申请中国发明专利3项，授权1项（专利号：ZL201610125205.1）。作为通讯作者，已在高水平学术期刊如Cancer Lett，Eur. J. Med. Chem.等发表相关学术论文21篇，其中，16篇被SCI收载，含中科院一区论文5篇（IF>5），最高IF值为7.36；在全国药化会上做分会学术报告1次。已培养相关方向博士生3人，硕士生9人，10人次以上获得辽宁省或校级优秀硕士学位论文、省优秀毕业生及国家奖学金等荣誉。课题负责人近3年来获评辽宁省科技创新领军人才（特聘教授）、省优秀硕士学位论文指导教师、省学位与研究生教育先进个人等诸多荣誉称号；获评沈阳市自然科学学术成果一等奖、三等奖各1项。.